In this study, we isolated three acidic polysaccharide components (IAPS-1, IAPS-2, and IAPS-3) from Rough-haired Holly (Ilex asprella) roots and systematically evaluated their immunomodulatory activity in murine macrophages. The polysaccharides were structurally characterized using integrated chromatographic and spectroscopic techniques. Subsequent bioactivity screening revealed that IAPS-1 demonstrated stronger immunomodulatory effects compared to IAPS-2 and IAPS-3. Mechanistically, in vitro experiments showed that IAPS-1 significantly upregulated TLR4-mediated expression of pro-inflammatory cytokines in RAW 264.7 macrophages. Furthermore, in cyclophosphamide-induced immunosuppressed mice, IAPS-1 administration restored immune organ indices and elevated serum levels of critical inflammatory mediators. Collectively, our findings identify IAPS-1 as a well-defined composition polysaccharide with immunomodulatory activity via TLR4 signaling, highlighting its potential as a therapeutic candidate for immune dysfunction.